1. Field of the Invention
The present invention relates to fields of protein biology and diagnostics. More particularly, the present invention relates to improved muteins of streptavidin that specifically yet reversibly bind biotin.
2. Description of Related Art
Wild-type streptavidin is a tetrameric protein with four identical subunits. Two dimers self-associate to form a tetrameric structure. Each subunit can bind one biotin tightly with a dissociation constant (KD) in the range of 10−13 to 10−14 M (Wilchek and Bayer, 1990). This binding is considered to be irreversible and streptavidin has been applied to capture and immobilize biotinylated biomolecules. It is widely used in development of many diagnostic kits, biosensor chip, protein and DNA arrays and Western blot studies. However, wild-type streptavidin is not suitable for purification of biotinylated biomolecules. To extend its application, it would be ideal to develop engineered streptavidin muteins with reversible biotin binding ability so that these muteins can be applied to purify biotinylated molecules, to study protein-protein interactions (with one of the interacting proteins to be biotinylated) and to develop reusable biosensor chips and bioreactors (e.g., traditional bioreactors will have enzymes chemically immobilized. After many rounds of usage, bioreactors with the immobilized enzymes will become useless when the immobilized enzymes lose their activities. With the engineered streptavidin that can bind biotin in a reversible manner, one can immobilize these engineered streptavidin proteins to the bioreactor. The enzymes of interest can then be biotinylated and loaded to bioreactors with the immobilized streptavidin muteins to generate functional bioreactors. When the enzymes lose their activity, these inactive enzymes can be eluted off by biotin and the bioreactor can be reloaded with a new batch of biotinylated enzymes).
To develop streptavidin muteins with reversible binding ability, two approaches are common. The first approach is to replace one or more streptavidin amino acid residues that are critical in biotin binding with different residues. These changes can result in lowering the biotin binding affinity in these muteins (Qureshi et al., 2001; U.S. Pat. No. 6,312,916 B1). The second approach is to develop recombinant monomeric streptavidin (Wu and Wong, 2005a). This is based on the fact that a streptavidin subunit does not have a complete biotin binding pocket. A biotin binding pocket in subunit A requires a tryptophan 120 (Trp-120) residue from subunit D. This Trp-120 has been demonstrated to play an important role in biotin binding (Chilkoti et al., 1995).
A change of this residue to alanine (W120A) results in a streptavidin mutein (W120A) with a Kd of 3×10−9M for biotin (U.S. Pat. No. 6,312,916 B1). Furthermore, affinity matrices of monomeric avidin have been developed. The dissociation constant of biotin for monomeric avidin is reported to be ˜10−7M (Kohanski and Lane, 1990). The inventors reported the successful generation of engineered monomeric streptavidin (Wu and Wong, 2005a) via the recombinant DNA method. This monomeric streptavidin also has the biotin binding affinity (expressed in terms of the dissociation constant, Kd) in the range of 10−7M. However, each of the above-mentioned approaches has certain limitations, and thus improved muteins of streptavidin are needed.